CRYOPRESERVATION OF APICAL MERISTEMS OF WHITE CLOVER (TRIFOLIUM-REPENS L) BY VITRIFICATION

Apical meristems of white clover (Trifolium repens L.) were successfully cryopreserved by vitrification. Excised apical meristems from in vitro plantlets were precultured on agar-B5 medium containing 1.2 M sorbitol at 4-degrees-C for 2 days. Meristems following preculture were transferred to a 1.8 ml plastic cryotube and then a highly concentrated cryoprotective solution (designated PVS2) was added at 25-degrees-C or at 0-degrees-C. The meristems treated with PVS2 at 25-degrees-C for 5 min or at 0-degrees-C for 15 min were directly plunged into liquid nitrogen. After rapid warming in water at 25-degrees-C, the meristems were expelled into B5 medium containing 1.2 M sucrose. Meristems on filter paper over agar-B5 medium resumed growth within 3 days and directly developed shoots. The average rate of plant regeneration was about 80% in meristems treated with PVS2 at 25-degrees-C for 5 min or at 0-degrees-C for 15 min. This procedure for cryopreservation is very simple and appears promising as a routine method for cryopreserving apical meristems.